Flow cytometry is a technique wherein light (usually focused laser beams) of specific wavelengths is used to illuminate cells, beads, macromolecules, etc. as they flow in a narrow stream. Scattered light is collected in the forward and side directions to provide information relating to the size, structure, etc. of the particles.
A common practice in flow cytometry is to employ highly fluorescent dyes to label cells in specific ways. One common method is to attach these dyes to biologically active molecules such as antibodies which selectively attach themselves to specific sites on or inside of cells. In this way, cells having these binding sites will be labeled and demonstrate fluorescence of a particular emission color when illuminated by light of the appropriate wavelength.
Regardless of how these dyes find themselves associated with cells, the strength of the measured signal is to a first approximation proportional to the amount of dye present with the cell. This method of labeling cells is now widespread in the study of biological specimens and is routinely done in samples prepared for fluorescent microscopes, flow cytometers, or other applications.
Flow cytometers are unique among many of these instruments because they do not make aggregate measurements (signals coming from a number of cells). While fluorescent microscopy can also examine single cells, the process is usually characterized by small sample sizes, and poor quantitation.
It is common to use more than one fluorescent dye in an effort to gain additional information about the cells as they pass through the illuminating light beam. To distinguish the signals from each other, the fluorescent labels are typically chosen so that they can be excited by the same light source, but fluoresce at different colors which can be separated using one or more techniques. Most dyes have a range of wavelengths at which they can be excited from their ground state to excited states. These molecules emit light over a range of wavelengths which is red shifted with respect to the illumination source when they return to their ground state.
To more efficiently distinguish between different fluorescent light signals, the emission spectra of different dyes are chosen so there is little or no spectral overlap. A technique known as FRET (fluorescence resonance energy transfer) uses dye combinations to tune the emission spectra further away from the excitation light and thereby increase the range over which fluorescent signals can be distributed in the electromagnetic spectrum. Dyes that have narrower emissions are preferred to those with broad emissions since their use permits more dyes to be used at the same time. Armed with knowledge of the dyes' emission spectra, it is possible and practical to account or compensate for the influence of a spectrally adjacent dye on another dye's signal provided the emission overlap is not too great.
It is a common desire for flow cytometer practitioners to use several fluorescent labels at the same time. Rather than attempting to fit all these dyes along the electromagnetic spectrum without excessive emission overlap, a common technique is to use multiple light sources spatially separated along the stream of flowing cells. In this manner dyes that have the same emission spectra, but different excitation spectra can be used to label the cells and the signals can be more easily distinguished.
A common flow cytometer design is to collect the signal light from all spatially separated interrogation regions and use wavelength dependent mirrors to separate the differently colored light signals. Apertures can be used to pass the light coming from a single interrogation region. In this manner, each detector would be used to detect a specific wavelength range coming from a single interrogation region. Using this type of arrangement common in flow cytometry, it takes n detectors to measure n different wavelength signals.
The systems described above typically employ free space optics. Recently, some commercial flow cytometers have employed a linear array of optical fibers to separate light from different interrogation regions. The image of the flow cell is focused onto the input plane of a linear array of optical fibers. In this manner, light from each interrogation region enters a unique fiber.
Each of these fibers then delivers the light from a single interrogation region to an arrangement of wavelength dependent mirrors to direct light of different wavelength ranges each to a different detector. These arrangements of wavelength dependent mirrors are larger than desired (owing to the size of beam diameters, mirrors, and detector housings). Each of these arrangements require n detectors to measure n signals.
The wavelength dependent mirrors are arranged such that incident light is not normal to them, but rather at some other angle. To the extent that incident light is not normal, the spectral properties of the mirror change and more importantly, the s and p polarizations tend to have different reflection/transmission properties. Since the signal light in a flow cytometer is unpolarized, the transitions between reflected wavelengths and transmitted wavelengths become less sharp at off-normal incidence. To address this fact, manufacturers have tended to increase the path length between wavelength selective mirrors to approach a closer-to-normal angle of incidence. While effective, the system becomes larger in size and more sensitive to misalignments and airborne dust.